The objectives of this project are to: (1)\analyze the neutral glycosphingolipids and gangliosides from the leukocytes of normal donors and leukemic patients; (2)\find a model human leukemic cell line(s) to use as a model system to evaluate the regulation of neutral glycosphingolipids in human leukocytes; and (3)\determine if the glycosphingolipids of human leukocytes are I,i antigens. We have completed an initial structural characterization of the neutral glycosphingolipids and gangliosides of normal human leukocytes and leukemia cells. Most of this information was based on complete chemical and enzymatic analysis of the isolated and purified compounds. In the next 2 years, we plan to use comparative methods (the combined use of endo-\and exoglycosidases with high performance liquid chromatography) to allow us to evaluate the glycosphingolipids of a larger group of normal donors and leukemic patients. This will allow us to determine if there are significant variations in the glycosphingolipid compositions in the different populations of normal and leukemic leukocytes. We will also use a highly sensitive radiolabel, monoclonal antibody binding assay to determine the distribution of GD3 ganglioside (II3(NeuAc)2-LacCer) in normal and leukemic leukocytes. Thus far, we have found this ganglioside in the cells of two leukemic patients, but not in normal neutrophils or several other leukemic cell types. This assay will also be used to determine if long-chain neutral glycosphingolipids (more than four monosaccharides) of human leukocytes carry the I,i antigenic structures. These studies will aid in structural characterization of these minor, complex glycosphingolipids. To find an appropriate human leukemia cell line to serve as a model system to study the regulation of glycosphingolipid expression in leukocytes undergoing differentiation, we will characterize the 14C-labeled glycosphingolipids obtained from cells incubated with 14C-galactose. High performance liquid chromatography in combination with glycosidase treatment will be used to characterize the biosynthesized glycosphingolipids. A comparison of the glycosphingolipids synthesized by untreated cells and cells treated with differentiating agents (butyrate, dimethyl sulfoxide and phorbol diesters) will be made. The results of these analyses will enable us to determine the variations in glycosphingolipids among the different classes of human leukemia cells (lymphoid and nonlymphoid; T, B and null cells), to make comparisons with those of normal leukocytes and to determine whether unique glycosphingolipids exist in leukemic cells.